Solid phytase compositions stabilized with corn steep liquor

ABSTRACT

The present invention relates to solid enzyme, in particular phytase, compositions stabilized with a lactic acid source such as Corn Steep Liquor (CSL), and methods of producing the same. Preferred compositions additionally comprise starch and disaccharides such as lactose or trehalose.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of Ser. No. 10/618,477 filed on Jul.11, 2003, now abandoned, which is a continuation of U.S. applicationSer. No. 09/410,503 filed Oct. 1, 1999 now U.S. Pat. No. 6,610,519,which issued as U.S. Pat. No. 6,610,519, which claims priority or thebenefit under 35 U.S.C. 119 of Danish application no. PA 1998 01251filed on Oct. 2, 1998 and U.S. provisional application No. 60/103,522filed on Oct. 8, 1998, the contents of which are fully incorporated byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to solid phytase compositions which havebeen stabilized with a lactic acid source such as Corn Steep Liquor(CSL), and methods of producing the same.

2. Description of Related Art

The addition of phytase to animal feed to eliminate the anti-nutritionaleffects of phytic acid is well described, see, e.g., WO 98/28408 and WO98/28409.

The stabilization of liquid phytase formulations with urea, glycerol orsorbitol is disclosed in WO 93/16175.

Salt-stabilized solid phytase compositions are disclosed in EP 0 758 018A1.

Plant seeds, cereal grains and legumes are usual components of animalfeed. Some of those seeds contain phytic acid, and often also endogenousphytase enzymes.

According to investigations performed by the applicant, endogenousphytase activity in animal feed is at a very low level of around 0.5units/g.

According to e.g., the two above first-cited WO-references, whensupplementary phytase has been added to feed, the phytase activity inthe feed is in the range of 0.01-20 units/g.

SUMMARY OF THE INVENTION

The present invention relates to solid phytase compositions whichcomprise (a) an enzyme having phytase activity; and (b) a lactic acidsource, wherein the phytase activity of the composition is above 20units/g.

DETAILED DESCRIPTION OF THE INVENTION

In the present context, the expression “enzyme (or polypeptide) havingphytase activity” or “phytase” includes any enzyme capable of effectingthe liberation of inorganic phosphate from phytic acid or from any saltthereof (phytates).

Phytic acid is myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate (orfor short myo-inositol hexakisphosphate). In what follows, unlessotherwise indicated, the terms “phytic acid” and “phytate,” are usedsynonymously or at random.

In the present context, the term “units” means units of enzyme, inparticular phytase, activity. Any method for determining phytaseactivity can be used.

In a preferred embodiment, one unit of phytase activity is defined asthe amount of enzyme that liberates 1 micro mole inorganicortho-phosphate per min. under the following conditions: A pH which iswithin the range of +/−1 pH unit from the optimum pH of the actualenzyme; a temperature which is within the range of +/−20° C. from theoptimum temperature of the actual enzyme; using as a substrate phyticacid or any salt thereof in a suitable concentration.

Preferably, the substrate is dodeca-sodium phytate in a concentration of0.005 mole/l.

Preferably, the pH is within the range of +/−0.5 pH unit from theoptimum pH; more preferably the pH is the optimum pH.

Preferably, the temperature is within the range of +/−10° C. from theoptimum temperature; more preferably the temperature is the optimumtemperature.

Preferably, the optimum pH and optimum temperature refers to the use ofsodium phytate as a substrate.

In another preferred embodiment, the phytase activity is determined inthe unit of FYT, one FYT being the amount of enzyme that liberates 1micro mole inorganic ortho-phosphate per min. under the followingconditions: pH 5.5; temperature 37° C.; substrate: sodium phytate(C₆H₆O₂₄P₆Na₁₂) in a concentration of 0.0050 mole/l.

In a further preferred embodiment, the phytase activity is measuredusing the FTU assay.

The FYT- and FTU-assays are described in more detail in the experimentalpart.

In preferred embodiments, the phytase activity of the solid compositionof the invention is above 25, 50, 100, 250, 500, 750 or even above 1000units/g.

Optionally, the phytase activity of the solid composition is below100,000 units/g, more preferably below 75,000 units/g, even morepreferably below 50,000 units/g, or below 40,000 units/g, or below25,000 units/g, or even below 10,000 units/g, mostly preferred below5,000 units/g.

Preferred ranges of phytase activity are 25-100,000, 25-75,000,35-50,000, or 50-40,000 units/g; more preferably 100-25.000 units/g;even more preferably 500-10.000 units/g; mostly preferred 1000-5000units/g.

In the present context, any enzyme having phytase activity can be used.

Phytases have been derived from plants as well as from microorganisms.Amongst the microorganisms, phytase producing bacteria as well asphytase producing fungi are known. From the plant kingdom, e.g., awheat-bran phytase is known (Thomlinson et al., 1962, Biochemistry 1:166-171). An alkaline phytase from lilly pollen has been described byBarrientos et al., 1994, Plant. Physiol. 106: 1489-1495.

Amongst the bacteria, phytases have been described which are derivedfrom Bacillus subtilis (Paver and Jagannathan, 1982, Journal ofBacteriology 151: 1102-1108) and Pseudomonas (Cosgrove, 1970, AustralianJournal of Biological Sciences 23: 1207-1220). Still further, a phytasefrom E. coli has been purified and characterized by Greiner et al.,1993, Arch. Biochem. Biophys. 303:107-113).

Phytase producing yeasts are also described, such as Saccharomycescerevisiae (Nayini et al., 1984, Lebensmittel Wissenschaft undTechnologie 17: 24-26. However, this enzyme is probably a myo-inositolmonophosphatase (Wodzinski et al., Adv. Appl. Microbiol. 42: 263-303).AU-A-24840/95 describes the cloning and expression of a phytase of theyeast Schwanniomyces occidentalis.

There are several descriptions of phytase producing filamentous fungi,primarily belonging to the fungal phylum of Ascomycota (ascomycetes). Inparticular, there are several references to phytase producingascomycetes of the Aspergillus genus such as Aspergillus terreus (Yamadaet al., 1986, Agric. Biol. Chem. 322: 1275-1282). Also, the cloning andexpression of the phytase gene from Aspergillus niger var. awamori hasbeen described (Piddington et al., 1993, Gene 133: 55-62). EP 0 420 358describes the cloning and expression of a phytase of Aspergillus ficuum(niger). EP 0 684 313 describes the cloning and expression of phytasesof the ascomycetes Myceliophthora thermophila and Aspergillus terreus.

Phytases derived from fungi of the phylum Basidiomycota are disclosed inWO 98/28409 and WO 98/28408.

Modified phytases or phytase variants are obtainable by methods known inthe art, in particular by the methods disclosed in EP 0897010, EP0897985, PCT/DK99/00153 and PCT/DK99/00154. The phytases disclosed inthese four patent applications can also be used in the compositions ofthe present invention.

A solid or dry composition is a particulate material comprising,preferably consisting essentially of, or consisting of, freely flowingparticles of a size ranging from (micro-m) 0.01, or from 1.0, orpreferably from around 1 to 1000, or to 1200, or to 1500, or even up to2000 (micro-m).

Preferably, a solid or dry phytase composition is such composition whichcan be prepared from liquid phytase concentrates e.g., by spray drying,spray cooling (prilling), or any type of granulation.

For spray drying, no further components need to be added to the liquidphytase concentrate.

For spray cooling, a meltable component—such as palm oil (and/or anothermeltable vegetable oil or fat), hydrogenated palm oil (and/or anotherhydrogenated vegetable oil), tallow, hydrogenated tallow or a waxfunctions as a matrix. The phytase and other ingredients, if any, areintroduced into the melted, meltable component, and the melt is thenallowed to solidify under particle-forming conditions, typically in aspray drying tower.

For many uses, however, including the use in animal feed, granulates areusually preferred for a number of reasons. One reason is that they mayreadily be mixed with feed components, or more preferably, form acomponent of a pre-mix which contains other desired feed additives suchas vitamins and minerals.

The particle size of the enzyme granulates preferably is compatible withthat of the other components of the mixture. This provides a safe andconvenient mean of incorporating enzymes into, e.g., animal feed.

The size of a particle may be regarded as the greatest linear dimensionof the particle; thus, in the case of, e.g., a substantially sphericalparticle (such as a substantially spherical granulate particle), theparticle size in question will be the diameter of the particle.

Agglomeration granulates and agglomerated powders may be prepared usingagglomeration technique in a high shear mixer (e.g., Lödige) duringwhich one or more filler materials and the enzyme are co-agglomerated toform granules.

Absorption granulates are prepared by having cores of one or morecarrier materials to absorb/be coated by the enzyme.

Typical filler materials are salts such as di-sodium sulphate andcalcium-lignosulphonate. Other fillers are silica, gypsum, kaolin, talc,magnesium aluminium silicate and cellulose fibers. Optionally, binderssuch as dextrins are also included in agglomeration granulates.

Typical carrier materials may consist of particulate cores having asuitable particle size. The carrier may be water soluble or waterinsoluble, e.g., starch, e.g., in the form of cassava or wheat; or asugar (such as sucrose or lactose), or a salt (such as sodium chlorideor sodium sulphate).

Optionally, the granulates are coated with a coating mixture. Suchmixture comprises coating agents, preferably hydrophobic coating agents,such as hydrogenated palm oil and beef tallow, and if desired otheradditives, such as calcium carbonate or kaolin.

WO 97/39116 discloses preferred processes for making solid compositionsof the invention in the form of enzyme-containing granules or anenzyme-containing granulate, see in particular the sections of thedetailed description therein headed cores, binders, fillers,plasticizers, fibrous materials, superabsorbents, coating layers,enzymes, other adjunct ingredients (these sections being herebyincorporated by reference herein). However, WO 97/39116 does notdisclose the inclusion in the solid composition of a lactic acid source.

Preferred methods of preparing phytase granulates are referred to inExample 3.

Preferred solid compositions of the invention are enzyme compositions.The preferred compositions are concentrated, viz. of an activity ofabove 20 units/g. Thus, the concept of solid enzyme compositioncomprises in particular, but are not limited to, spray-dried enzymepreparations, enzyme granulates, e.g., agglomeration granulates andabsorption granulates, coated as well as un-coated, andenzyme-containing pre-mixes for animal feed. Phytase is a preferredenzyme.

Liquid enzyme (phytase) concentrates can e.g., be prepared as follows:The enzyme source, typically a phytase-containing fermentation broth, issubjected to a primary separation step (e.g., using a decanter, acentrifuge, or a filter press), followed by a second polish filtrationand/or germ filtration step. Finally the liquid is concentrated, e.g.,using ultra filtration, followed by a germ filtration. A typical drymatter content is in the range of 10-30%, preferably 15-25%, morepreferably 17-22%.

In the present context, “a” generally means “one or more” or “at leastone.” This applies, inter alia, for the following mandatory or optionalcomponents of the compositions of the invention: Phytase, lactic acidsource, CSL, starch, disaccharide, filler, and carrier.

Unless otherwise indicated, all percentage indications areweight/weight, by reference to dry matter content. Preferably, “units/g”also refers to dry matter content. Dry matter content can be determinedby any method known in the art, such as refractometer or drying in anoven to release humidity.

Unless otherwise indicated, the expression “above” generally means “≧”,whereas the expressions “up to” or “below” mean “≦”.

In the present context a “lactic acid source” or a “lactic acidpreparation” is any composition which comprises the compound lactic acidor any lactates, i.e., any salts thereof (lactic acid is 2-hydroxypropanic acid). Likewise, “lactic acid” as used herein includes anylactates. These expressions are used interchangeably for the lactic acidsource, resp. the lactic acid, as is, and for the dry matter partthereof.

A non-limiting list of lactic acid sources is the following: Lactic acidand lactates as relatively pure chemical compounds (purity of, say,above 70%, 80%, 90%); lactic acid and lactates as more impure substances(purity of, say, above 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%); anynatural or synthetical composition which comprises lactic acid in anamount of above 5%, preferably above 10%, 15%, 20%, 25%, 30%, 40%, 50%,70%, 80%, 90%.

The solid enzyme compositions of the invention preferably comprise up to20, preferably up to 15, more preferably up to 10, still more preferablyup to 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.75 or 0.5% lactic acid. The contentof lactic acid is preferably above 0.001, preferably above 0.002, 0.004,0.006, 0.008, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.12, 0.14, 0.16, 0.18,0.2, 0.22, 0.24, 0.26, 0.28, or above 0.3%. Preferred ranges of contentof lactic acid are 0.01-10%, 0.02-9%, 0.03-8%, 0.04-7%, 0.05-6%,0.06-7%, 0.07-6%, 0.08-5%, 0.09-4%, or 0.1-3%.

Any assay for lactic acid can be used. Preferred lactic acid assays arefrom SIGMA: (1) Assay kit catalogue no. 735-10 (enzymatical assay,lactate degraded to pyruvate and hydrogen peroxide in the presence ofoxidase); or (2) Assay kit catalogue no. 826-A and 826-B (ultraviolet,endpoint, lactate converted into pyruvic acid in the presence of lactatedehydrogenase and NAD).

A preferred lactic acid source is Corn Steep Liquor or CSL. CSL is acommercially available product, see for instance Merck Index, 1996,4^(th) edition, Index no. 2598. It is a viscous yellowish or dense brownliquid obtained by concentration of corn steep water. The dry mattercontent is usually 45-55%, preferably 48-52%. The pH is in the range of3-5, preferably 3.5-4.5. The protein content (Dry Matter) is typically30-50%, preferably 35-45%. The acidity (as lactic acid) is typically10-30% (Dry Matter), preferably 12-25%.

“CSL” as used herein refers to the product as such, or to its dry matterpart.

In a preferred embodiment, the solid composition of the inventioncomprises 0.01-15%, preferably 0.1-10%, more preferably 1-5% of CSL.

For analyzing CSL-content, any method can be used. A preferred HPLCmethod for fingerprinting and quantifying CSL is indicated in Example 8.Another preferred method is Head Space Gas Chromatograph (HS-GC),preferably combined with mass spectrometry (MS).

In a preferred embodiment, the solid composition of the inventionadditionally comprises a starch source, typically in an amount of0.1-20%, more preferably 0.2-10%, still more preferably 1-5%.

The concept of a starch source includes any natural or syntheticpolysaccharides comprising glucose units interconnected by alpha-1,4 oralpha-1,6-linkages. Purity is preferably above 10, 20, 30, 40, 50, 60,70, 80, 90, or 100%. A preferred starch source is Wheat Starch, which isa commercially available product. The expression “starch source”includes the starches and modified starches described in the sectionheaded “Cores” of WO 97/39116, cited above.

In another preferred embodiment, the solid composition of the inventionadditionally comprises a disaccharide, preferably in an amount of0.01-15%, even more preferred 0.1-10%, even more preferably 1-5%.

The concept of disaccharides includes any natural or syntheticdisaccharides, whatever the monomers, and whatever the linkage type.Examples of such disaccharides are maltose, lactose, cellobiose,sucrose, and trehalose (non-limiting list). Preferably, thedisaccharides are of a purity of above 10, 20, 30, 40, 50, 60, 70, 80 oreven 90%. Preferred disaccharides are lactose and trehalose(alpha-D-glucose alpha-D-glucopyranoside, alpha-1,1 linkage).

In the process of the invention, all steps, e.g., those indicated inclaim 14, can be performed simultaneously or sequentially. For example,steps (i) and (ii) sequentially or preferably simultaneously (mixing thelactic acid source and the phytase before spraying it onto the carrier);steps (iii) and (iv) simultaneously or sequentially, preferablysimultaneously, in the same apparatus; applies also to “together with”of claim 12.

Further preferred embodiments of the invention are the following:

A solid composition which comprises at least one enzyme having phytaseactivity, and Corn Steep Liquor (CSL), wherein the phytase activity ofthe composition is in the range of 20-50.000 units/g. A preferred amountof CSL is within the range of 0.01-15% (dry matter content and w/w).Preferably, the composition additionally comprises Wheat Starch (WS),preferably in an amount within the range of 0.01-20% (dry matter contentand w/w);

A process for preparing a granulate composition having a phytaseactivity in the range of 20-50.000 units/g, which method comprises thesteps of (i) spraying a liquid phytase concentrate onto a carrier; (ii)spraying CSL onto the carrier; (iii) mixing; and (iv) drying;

A process for preparing a spray dried solid composition having a phytaseactivity in the range of 20-50.000 units/g, which method comprises thestep of adding CSL to a liquid phytase concentrate before spray dryingit.

The activities of the solid phytase compositions prepared in the presentexamples are in the range of 1000-3000 FTU/g before storage.

Example 1 FYT Assay

10 microliters diluted enzyme samples (diluted in 0.1 M sodium acetate,0.01% Tween 20, pH 5.5) are added into 250 microliters 5 mM sodiumphytate (Sigma) in 0.1 M sodium acetate, 0.01% Tween 20, pH 5.5 (pHadjusted after dissolving the sodium phytate; the substrate ispreheated) and incubated for 30 minutes at 37° C. The reaction isstopped by adding 250 microliters 10% TCA and free phosphate is measuredby adding 500 microliters 7.3 g FeSO4 in 100 ml molybdate reagent (2.5 g(NH₄)₆Mo₇0₂₄.4H₂0 in 8 ml H₂SO₄ diluted to 250 ml). The absorbance at750 nm is measured on 200 microliter samples in 96 well microtiterplates. Substrate and enzyme blanks are included. A phosphate standardcurve is also included (0-2 mM phosphate). 1 FYT equals the amount ofenzyme that releases 1 micromol phosphate/min at the given conditions.

FTU Assay

One FTU is defined as the amount of enzyme, which at standard conditions(37° C., pH 5.5; reaction time 60 minutes and start concentration ofphytic acid 5 mM) releases phosphate equivalent to 1 micromol phosphateper minute.1 FTU=1 FYT

The FTU assay is preferred for phytase activity measurements on animalfeed premixes and the like complex compositions.

Reagents/Substrates

Extraction Buffer for Feed Etc.

This buffer is also used for preparation of PO₄-standards and furtherdilution of premix samples.

0.22 M Acetate Buffer with Tween 20 pH 5.5

30 g sodium acetate trihydrate (MW=136.08 g/mol) e.g., Merck Art 46267per liter and 0.1 g Tween 20, e.g., Merck Art 22184 pr. liter areweighed out.

The sodium acetate is dissolved in demineralized water.

Tween 20 is added, and the pH is adjusted to 5.50±0.05 with acetic acid.

Add demineralized water to total volume.

Extraction Buffer for Premix

0.22 M acetate buffer with Tween 20, EDTA, PO₄ ³⁻ and BSA.

30 g sodium acetate trihydrate e.g., Merck Art 6267 per liter.

0.1 g Tween 20 e.g., Merck Art 22184 per liter.

30 g EDTA f.eks. Merck Art 8418 per liter.

20 g Na₂HPO₄, 2H₂O e.g., Merck Art 6580 per liter.

0.5 g BSA (Bovine Serum Albumine, e.g., Sigma Art A-9647 per liter.

The ingredients are dissolved in demineralized water, and pH is adjustedto 5.50±0.05 with acetic acid.

Add demineralized water to total volume.

BSA is not stable, and must therefore be added the same day the bufferis used.

50 mM PO₄ ³⁻ Stock Solution

0.681 g KH₂PO₄ (MW=136.09 g/mol) e.g., Merck Art 4873 is weighed out anddissolved in 100 ml 0.22 M sodium acetate with Tween, pH 5.5.

Storage stability: 1 week in refrigerator.

0.22 M Acetate Buffer pH 5.5 without Tween

This buffer is used for production of phytic acid substrate).

150 g sodium acetate trihydrate (MW=136.08) e.g., Merck Art 6267 isweighed out and dissolved in demineralized water, and pH is adjustedwith acetic acid to 5.50±0.05.

Add demineralized water to 5000 ml.

Storage stability: 1 week at room temperature.

Phytic Acid Substrate; 5 mM Phytic Acid

The volume of phytic acid is calculated with allowance for the watercontent of the used batch.

If the water content is e.g., 8.4% the following is obtained:(0.005 mol/l×923.8 g/mol)/(1/0.084)=5.04 g/l

Phytic acid (Na-salt) (MW=923.8 g/mol) e.g., Sigma P-8810 is weighed outand dissolved in 0.22 M acetate buffer (without Tween). Addition of(diluted) acetic acid increases the dissolution speed.

The pH is adjusted to 5.50±0.05 with acetic acid.

Add 0.22 M acetate buffer to total volume.

21.7% Nitric Acid Solution

For stop solution.

1 part concentrated (65%) nitric acid is mixed into 2 partsdemineralized water.

Molybdate Reagent

For stop solution.

100 g ammonium heptamolybdate tetrahydrate (NH₄)₆Mo₇O₂₄, 4H₂O, e.g.,Merck Art 1182 is dissolved in demineralized water. 10 ml 25% NH₃ isadded.

Add demineralized water to 1 liter.

0.24% Ammonium Vanadate

Bought from Bie & Berntsen.

Molybdate/Vanadate Stop Solution

1 part vanadate solution (0.24% ammonium vanadate)+1 part molybdatesolution are mixed. 2 parts 21.7% nitric acid solution are added.

The solution is prepared not more than 2 hours before use, and thebottle is wrapped in tin foil.

Samples

Frozen samples are defrosted in a refrigerator overnight.

Sample size for feed samples: At least 70 g, preferably 100 g.

Feed Samples

Choose a solution volume which allows addition of buffer correspondingto 10 times the sample weight, e.g., 100 g is dissolved in 1000 ml 0.22M acetate buffer with Tween, see enclosure 1. Round up to nearestsolution volume.

If the sample size is approx. 100 g all the sample is ground in a coffeegrinder and subsequently placed in tared beakers. The sample weight isnoted. It is not necessary to grind not-pelleted samples. If a sample istoo big to handle, it is sample split into parts of approx. 100 g.

Magnets are placed in the beakers and 0.22 M acetate buffer with Tweenis added.

The samples are extracted for 90 minutes.

After extraction the samples rest for 30 minutes to allow for the feedto sediment. A 5 ml sample is withdrawn with a pipette. The sample istaken 2-5 cm under the surface of the solution and placed in acentrifuge glass, which is covered by a lid.

The samples are centrifuged for 10 minutes at 4000 rpm.

Premix Samples

Choose a solution volume which allows addition of buffer correspondingto 10 times the sample weight. Round up to nearest solution volume.

If the samples have been weighed (50-100 g) all of the samples areplaced in tared beakers. The sample weight is noted. If a sample is toobig to handle, it is split into parts of approx. 100 g.

Magnets are placed in the beakers and 0.22 M acetate buffer with Tween,EDTA and PO₄ ³⁻ is added.

The samples are extracted for 60 minutes.

After extraction the samples rest for 30 minutes to allow for the premixto sediment. A 5 ml sample is withdrawn with a pipette. The sample istaken 2-5 cm under the surface of the solution and placed in acentrifuge glass, which is covered by a lid.

The samples are centrifuged for 10 minutes at 4000 rpm.

Analysis

Extracts of feed samples are analyzed directly.

Extracts of premix are diluted to approx. 1.5 FTU/g (A₄₁₅ (mainsample)<1.0).

0.22 M acetate buffer with Tween 20 is used for the dilution.

Main Samples

2×100 ml of the supernatant from the extracted and centrifuged samplesare placed in marked glass test tubes and a magnet is placed in eachtube.

When all samples are ready they are placed on a water bath withstirring. Temperature: 37° C.

3.0 ml substrate is added.

Incubation for exactly 60 minutes after addition of substrate.

The samples are taken off the water bath and 2.0 ml stop solution isadded (exactly 60 minutes after addition of substrate).

The samples are stirred for 1 minute or longer.

Feed samples are centrifuged for 10 minutes at 4000 rpm (It is notnecessary to centrifuge premix samples).

Blind Samples

100 ml of the supernatant from the extracted and centrifuged samples areplaced in marked glass test tubes, and a magnet is placed in each tube.

2.0 ml stop solution is added to the samples.

3.0 ml substrate is added to the samples.

The samples are incubated for 60 minutes at room temperature.

The feed samples are centrifuged for 10 minutes at 4000 rpm (it is notnecessary to centrifuge premix samples).

Standards

2×100 ml are taken from each of the 8 standards and also 4×100 ml 0.22 Macetate buffer (reagent blind).

A₄₁₅ is measured on all samples.

CalculationFTU/g=micromol PO₄ ³⁻/(min*g(sample))

C g sample is weighed out (after grinding).

100 microliters are taken from the extracted and centrifuged sample.

PO₄ ³⁻ standard curve is linear.

From the regression curve for the PO₄ ³⁻ standard the actualconcentration of the sample is found (concentration in mM):[PO₄ ³⁻]=(x−b)/a x=A ₄₁₅ a=slope b=intercept with y-axismicromol PO₄ ³⁻/min={[PO₄ ³⁻](mM)×Vol(liter)×1000 micromol/mmol}/tt=incubation time in minutes.Vol=sample volume in liter=0.0001 liter1000=conversion factor from mmol to micromolFTU/g_(prøve)={(x−b)×Vol×1000×F _(p) }/{a×t×C}C=gram sample weighed outF_(p)=Relation between the sample taken out and the total sample (afterextraction).Example: 0.100 ml taken from 1000 ml→F_(p)=1000/0.100=10000.Reduced expression with insertion of the following values:t=60Vol=0.0001|F_(p)=10000FTU/g_(prøve)={(x−b)×0.0001×1000×10000}/{a×60×C}

Example 2 Preparation of a Concentrated Liquid Phytase Preparation

The phytase derived from Peniophora lycii is expressed in Aspergillusoryzae, fermented and purified, essentially as described in WO 98/28408.The resulting liquid phytase concentrate is a UF (ultra filtration)concentrate of a dry matter content of 18%. The pH is adjusted to 5.

Example 3 Preparation of Phytase Granulates

A coated phytase granulate with 1.5% CSL is prepared as follows:

14.68 kg of a powder composition with the formulation

0.75 kg kaolin, Speswhite, English China Clay

1.80 kg of fibrous cellulose, Arbocel BC 200

11.23 kg finely ground sodium sulphate

0.90 kg Carbohydrate binder, Tackidex G155 from Roquette

is mixed in a Lödige mixer FM 50 and sprayed with 3.15 kg of a sprayingliquid consisting of 1.68 kg of water, 0.625 kg of Corn Steep Liquor(Concentrated Corn Steep Liquor (CCSL) supplied by Amylum N.V. with adry matter content of 48%) and 0.84 kg of Phytase concentrate (18% drymatter content) prepared as described in Example 2. During and afterspraying the moist mixture is exposed to a compacting and granulationinfluence from the multiple set of knives, as described in Example 1 ofU.S. Pat. No. 4,106,991.

The percentage of CSL in this as yet un-coated raw granulate iscalculated as follows:0.625×0.48/(14.68+0.625×0.48+0.84×0.18)=0.300/(14.68+0.30+0.672)=0.300/15.652=1.917%˜2%.

The granulate is dried in a fluid bed to a water content below 3%,resulting in a light coloured granulate with the following particledistribution:

10.5%>1100 micro-meter

92.0%>300 micro-m

8.0%<300 micro-m

The granulate is finally sifted to get a product with the particle range300 micro-m to 1100 micro-m, and 6 kg of granulate is coated at 80° C.with 9% fully hydrogenated palm oil, followed by 22.5% of kaolin,Speswhite (dry matter content in 100 g coating material: 22.5 g+9 g=31.5g), in a manner as described in U.S. Pat. No. 4,106,991, Example 22.

The content of CSL in the resulting final product, the coated granulate,is reduced as compared to the CSL content of the raw granulate asfollows: 1.917%/1.315=1.458%˜1.5%.

The granulate is sifted to obtain a product with the particle range 300micro-m to 1200 micro-m.

The control granulate used below is prepared as described above, exceptfor no CSL being added.

Granulates additionally comprising wheat starch and lactose or trehaloseare prepared in a corresponding manner.

Example 4 Storage Stability of Phytase Granulates in Premix

The phytase granulates indicated in Table 1 below are prepared accordingto Example 3. “Control” indicates a phytase granulate prepared accordingto the method of Example 3, but with no addition of CSL.

The granulates are weighed directly into each vial. The exact weight ofthe granulate is recorded. The vials are covered with a clean towel andleft at room temperature overnight.

The premix ENGA 1-02/Nordkorn. Product. No: 015384 Artikel Nr. 8259 (25kg drums) is mixed in a Lödige mixer to ensure an even distribution ofthe premix components and filled into plastic bags with ≈3 kg premix ineach bag.

The composition of the premix is as follows (per kilo):

5000000 IE Vitamin A 1000000 IE Vitamin D3 13333 mg Vitamin E 1000 mgVitamin K3 750 mg Vitamin B1 2500 mg Vitamin B2 1500 mg Vitamin B6 7666mcg Vitamin B12 12333 mg Niacin 33333 mcg Biotin 300 mg Folic Acid 3000mg Ca-D-Panthothenate 1666 mg Cu 16666 mg Fe 16666 mg Zn 23333 mg Mn 133mg Co 66 mg I 66 mg Se 5.8% Calcium 25% Sodium

50 g±1 g of premix is added to each vial and the vials are closed with ascrew-on lid. The premix is added using an adjustable cylindrical“scoop” adjusted to give a volume corresponding to 50 g. The vials aremixed by hand until the granulates are evenly distributed in the premix.

The 0 week samples (closed vials), defining for each granulate the levelof 100% activity, are frozen immediately after completion of the samplepreparation. The samples which are to be stored at 30° C. are re-opened.The open vials are placed in plastic boxes containing 1 liter ofglycerol adjusted with water to 43% rH (62% refractometer dry mattermeasured on a sugar scale) corresponding to ≈10% water in the samples.The lids of the plastic boxes are sealed with strong tape. This meansthat the water activity is 0.43 during the whole test period of 13weeks.

After completion of the storage period the samples are removed from theglycerol boxes, closed with screw on lids and frozen.

The samples are defrosted in a refrigerator (5° C.) overnight prior toanalysis.

The 0 week samples stored at −18° C. and the corresponding samplesstored at 30° C. are analysed the same day in order to eliminateday-to-day and person-to-person variation.

The results are shown in Table 1 below; CSL=Corn Steep Liquor andWS=Wheat Starch.

Phytase granulates containing 2% CSL, 3% CSL, and 2% CSL plus 5% WSshowed similar performance.

TABLE 1 Percentage residual activity following 13 weeks storage atGranulate code Granulate 30° C. 1 Control granulate 61% 2 Controlgranulate 64% 3 1.5% CSL 81% 4 1.5% CSL 86% 5 1.5% CSL 84% 6 1.5% CSL85% 7 1.5% CSL + 3.8% WS 84% 8 1.5% CSL + 3.8% WS 90% 9 1.5% CSL + 3.8%WS 84%

Example 5 Storage Stability of Phytase Granulates in Feed

The phytase granulates indicated in Table 2 below are prepared accordingto Example 3. “Control” indicates a phytase granulate prepared accordingto the usual standard method of Example 3, except for neither WS nor CSLnor disaccharides being added.

The samples of granulates in feed are prepared at BioteknologiskInstitut, Kolding, Denmark.

The composition of the feed is as follows:

74.0% wheat

20.7% roasted soy cake

5.0% soy oil

0.3% Premix Enga 1-02/Nordkorn

The feed is dried to a water content of ≦10% water before addition ofthe phytase granulates.

The granulate batches are mixed into feed and the mixture is pelletizedat 65° C.

The feed pellets are sample split and filled into 100 ml sample vials.

The 0 week samples, defining for each granulate the level of 100%activity, are closed with screw on lids and kept at −18° C.

The samples which are to be stored at 30° C. are not closed. The openvials are placed in plastic boxes containing 1 liter of glyceroladjusted with water to 43% rH (62% refractometer dry matter measured ona sugar scale) corresponding to ≈10% water in the samples. The lids ofthe plastic boxes are sealed with strong tape. This means that the wateractivity is 0.43 during the whole test period of 13 weeks.

After completion of the storage period the samples are removed from theglycerol boxes, closed with screw on lids and frozen. The samples aredefrosted in a refrigerator (5° C.) overnight prior to analysis.

Samples for the homogeneity test are kept refrigerated at +5° C. untilanalysis.

The mash feed, the feed heated to 65° C., and the feed pellets withoutadded enzyme all contain ≈0.5 FTU/g feed as expected.

5 samples of the mash feed with enzyme added and heated to 65° C. areanalysed for homogeneity. The relative standard deviation is 2% to 11%.In conclusion, the homogeneity is acceptable.

5 samples of the feed pellets are analysed for homogeneity. The relativestandard deviation is 2% to 10%. In conclusion, the homogeneity isacceptable.

The storage stability is measured after 13 weeks. The 0 week samplesstored at −18° C. and the corresponding samples stored at 30° C. areanalyzed the same day in order to eliminate day-to-day andperson-to-person variation.

The results of the phytase residual activity measurements are shown inTable 2 below (endogenous activity has been subtracted from the totalactivity before calculating the residual activity); CSL=Corn SteepLiquor and WS=Wheat Starch.

Phytase granulates containing 2% CSL, 3% CSL, and 2% CSL plus 5% WSshowed similar performance.

TABLE 2 Percentage residual activity following 13 weeks storage atGranulate code Granulate 30° C. 1 Control granulate 53% 2 Controlgranulate 55% 3 1.5% CSL 74% 4 1.5% CSL 89% 5 1.5% CSL 82% 6 1.5% CSL73% 7 1.5% CSL + 3.8% WS 82% 8 1.5% CSL + 3.8% WS 91% 9 1.5% CSL + 3.8%WS 89%

Example 6 Phytase Granulates Per Se; Granulation Yield and StorageStability

The liquid concentrate of Example 2 was used to prepare experimentalsolid phytase compositions according to the method of Example 3.

In a first granulation experiment, the disaccharide lactose was added inan amount of 2% together with 3% of the lactic acid source Corn SteepLiquor (CSL).

In a second granulation experiment, 3% Wheat Starch (WS) was applied—inaddition to the two components of the first experiment.

In a third granulation experiment, the disaccharide trehalose, in anamount of 2%, as well as 3% WS, was added together with 3% of the lacticacid source CSL.

The effect on granulation yield and storage stability of the resultinggranulates per se is examined.

Granulation yield is calculated as phytase units remaining in theproduct leaving the granulation unit, relative to phytase units of theliquid concentrate entering the unit.

The storage stability of the resulting phytase granulate composition perse is examined using the following rather strict conditions: 4 weeks,40° C. and a relative humidity of 60%.

The results are shown in Table 3 below.

TABLE 3 Granulation yield Storage stability Experiment Batch 22 Batch 24Batch 22 Batch 24 Control 75% 70% 43% 47% CSL + lactose 80% 67% 47% 50%CSL + WS + lactose 81% 79% 56% 54% CSL + WS + trehalose 85% 82% 63% 62%

Example 7 Storage Stability of a Granulate Composition of AnotherPhytase

A liquid phytase concentrate and a solid composition—i.e., agranulate—was prepared according to the teachings of Examples 2 and 3,using a so-called consensus phytase as described in EP 0897010.

Granulation experiments were conducted essentially as described inExample 6. However, for storage stability samples are also stored at 30°C. The results are shown in Table 4 below.

TABLE 4 Storage stability 40° C., Granulation 30° C. 60% RH Experimentyield 8 weeks 17 weeks 4 weeks Control 82% 85% 85% 40% CSL + WS +lactose 92% 97% 95% 48%

Example 8 Characterization of CSL Using High Performance LiquidChromatography (HPLC)

15 samples from various batches of CSL from various suppliers (RoquetteFreres, 4 Rue Patou, F-59022 Lille Cedex, France; Staral s.a., Z.I.ETPortuaire, B.P. 32, F-67390 Marckolsheim, France; and CerestarScandinavia A/S, Skovlytoften 33, DK-2840 Holte, Denmark) are tested asdescribed below.

Carrez-Precipitation

Weigh 5.0 g CSL into a 100 ml flask. Add 40 ml MQ-water (demineralizedwater filtered through a Milli-q filter) and incubate at 70° C. for 15minutes while shaking at 200 rpm. Add 12 ml of Carrez-I-solution(Potassium-hexacyanoferrat(II)-tri hydrate) and shake. Add 12 mlCarrez-II-solution (Zinc sulphate-hepta hydrate) and shake. Add 20 ml0.5N NaOH and shake. Let cool and add MQ-water ad 100 ml, shake. 10 mlof this preparation is transferred to a vial and centrifuged for 10minutes at 4000 rpm. The supernatant is filtered at a 0.5 micro-m filterfor HPLC analysis. Each sample is analyzed twice, include sample blinds(MQ-water and Carrez-solutions).

Chromatography Parameters

Column: Supelcosil LC-18-DB, No. 088877AE

Detector: Shimadzu SPDM6A-diodearray from 220 nm to 350 nm.

Data analysis: For analyzing data, use peak areas resulting fromintegration at 260 nm.

Pump: HP 1080 gradient pump

Eluents: A) MQ-water

-   -   B) 30% MeOH    -   C) 60% MeOH    -   D) 90% MeOH

Gradients: 0 min A

-   -   15 min A    -   35 min B    -   50 min C    -   60 min C    -   65 min D    -   70 min D    -   75 min C    -   80 min B    -   85 min A    -   90 min A

The results of variable statistics on 15 samples of CSL analyzed by HPLCare shown in Table 5 below:

TABLE 5 Standard Peak Deviation retention time Min Max Mean (SD) % SD6791 22154 114227 79192 25978 32.80⁷ 7445 1934348 2326644 2135867 1109115.19¹ 10331 4746 132048 44503 52860 118.78 11844 0 3020 201 780 387.3012440 124723 183826 155946 17166 11.01⁴ 14124 79613 186731 119578 3503229.30⁶ 15322 0 27204 13205 11537 87.37 16187 314623 380627 346898 212716.13² 18831 18494 148325 130989 10481 8.00³ 26373 0 8482 2288 3454150.99 26833 0 59860 25622 19252 75.14 27672 0 46259 24388 13200 54.1228053 0 11383 3085 3169 102.71 28762 5643 60078 31762 20966 66.01 294913657 14650 8970 3353 37.38⁸ 29926 40184 89538 52754 14807 28.07⁵ 30607 044749 14732 15523 105.37 30951 0 19732 2423 5786 238.80 31825 0 160902624 4909 187.06 32454 0 15725 1288 4050 314.36 32636 0 28268 7516 9534126.85 33068 0 36398 4570 9707 212.42 33394 0 96671 30197 33359 110.4733646 0 24856 4042 7600 188.02 34108 0 11826 2144 3922 182.94 34464 029248 9794 8990 91.79 35309 0 14392 8793 6491 73.82 36826 0 29619 966710423 107.82 42457 0 45570 32023 12404 38.73⁹ 42971 29905 102074 4530318156 40.08¹⁰ 43427 0 49318 6293 14306 227.33 43812 0 13837 5851 492484.16 45519 0 12487 3570 4815 134.87 46032 0 11850 4728 4756 100.5946654 0 36357 23766 11561 48.65 47034 0 37769 17540 11212 63.92 47268 020271 5414 6969 128.72 47784 0 5218 1569 2091 133.25 48494 0 5858 18721595 85.20 48859 0 10935 2923 3748 128.25 49180 0 24091 11016 8573 77.8249467 0 23885 10146 8372 82.52 49905 0 22011 4205 8506 202.31

In the % SD column of Table 5, characteristic peaks are indicated by wayof a superscript number (1, 2, 3, - - - , 9, 10). In what follows, thesepeaks will be referred to as peak-1, peak-2, peak-3, - - - , peak-9,peak-10, respectively. The whole group of ten peaks is referred to aspeaks 1-10. Sub-groups are referred to by analogy, e.g., peaks 1-5 forthe five peaks numbered 1 to 5, peaks 1, 3, 5 for peak-1, peak-3 andpeak-5 etc. Thus, the presence of one or more of these peaks in a sampleis indicative of the presence of CSL. In preferred embodiments, thepresence of one, two, three, four, five, six, seven, eight, nine or allten of these peaks is indicative of the presence of CSL. In morepreferred embodiments, the presence of five, seven, eight or ten peaksis indicative of the presence of CSL. The presence of five peaks is mostpreferred.

For samples of an unknown content of CSL, suitable dilutions are foundusing simple trial-and-error techniques.

The above qualitative method can be made quantitative by comparing witha batch denominated by Roquette Freres to be a standard batch. Aparticularly preferred standard CSL batch from Roquette Freres isSOLULYS® L 48 L CAS No. 66071-94-1, EINECS: 266-113-4.

The invention claimed is:
 1. A solid phytase composition, comprising:(a) an enzyme having a phytase activity of above 20 FYT/g of thecomposition, and (b) corn steep liquor in an amount sufficient tostabilize the enzyme having phytase activity, wherein the corn steepliquor is present in an amount of 0.1-15%, and wherein the enzyme havingphytase activity in the composition has a residual activity of 73% to91% following 13 weeks storage at 30° C.
 2. The composition of claim 1,having a chromatogram determined by HPLC, which has one or more of peaks1-10.
 3. The composition of claim 1, further comprising a starchmaterial.
 4. The composition of claim 1, further comprising wheatstarch.
 5. The composition of claim 1, further comprising adisaccharide.
 6. The composition of claim 1, further comprising acarrier material.
 7. The composition of claim 1, further comprising afiller material.
 8. The composition of claim 1, further comprising oneor more vitamins, one or more minerals or a mixture of both.
 9. Thecomposition of claim 1, wherein the phytase activity is at least 25FYT/g of the composition.
 10. The composition of claim 9, wherein thephytase activity is at least 50 FYT/g of the composition.
 11. Thecomposition of claim 10, wherein the phytase activity is at least 100FYT/g of the composition.
 12. The composition of claim 11, wherein thephytase activity is at least 250 FYT/g of the composition.
 13. Thecomposition of claim 12, wherein the phytase activity is at least 500FYT/g of the composition.
 14. The composition of claim 13, wherein thephytase activity is at least 750 FYT/g of the composition.
 15. Thecomposition of claim 14, wherein the phytase activity is at least 1,000FYT/g of the composition.
 16. The composition of claim 1, wherein thephytase activity is in the range of 20-50,000 FYT/g.
 17. The compositionof claim 1, wherein the phytase activity is in the range of 100-25,000FYT/g.
 18. The composition of claim 1, wherein the phytase activity isin the range of 500-10,000 FYT/g.
 19. The composition of claim 1,wherein the phytase activity is in the range of 1,000-5,000 FYT/g. 20.The solid phytase composition of claim 1, wherein corn steep liquor ispresent in an amount of 0.1-10%.
 21. The solid phytase composition ofclaim 1, wherein corn steep liquor is present in an amount of 1-5%. 22.The solid phytase composition of claim 1, wherein corn steep liquor ispresent in an amount of about 1.5%.